We have found that the thiolester enantiomer (plus)-S-(trans-cinnamoyl)-alpha-mercapto-beta-phenylpropionate reacts with carboxypeptidase A by a different mechanism than does the corresponding (-)-thiolester enantiomer or the oxygen ester O-(trans-cinnamoyl)-L-beta-phenyllactate. This appears to be the first case in which it has been demonstrated that two enantiomers of the same substrate can react by different mechanistic pathways with the same enzyme. To obtain further information concerning the mechanisms involved, we plan to carry out the reaction of carboxypeptidase A modified in the Tyr-248 residue with the (plus)-thiolester and to pursue nucleophile trapping experiments on the reactions of a variety of metallocarboxypeptidases (the cadmium and mercury enzymes, for instance) with both ester and peptide substrates. Additionally, we have found strong evidence for the formation of an acyl-enzyme in the reaction of yeast carboxypeptidase, an exopeptidase which does not seem to contain an active site metal ion, with p-nitrophenyl trimethylacetate. We intend to probe the possibility that acyl-enzymes are involved in the exopeptidase activity of the enzyme and to determine the relationship between this type of activity and yeast carboxypeptidases nitrophenyl esterase action.